Native wharton&#39;s jelly stem cells and their purification

ABSTRACT

Noncultured Wharton&#39;s Jelly stem cells and methods of their purification, storage and use are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/701,329, filed Nov. 30, 2012, which is the U.S. national stage ofinternational patent application PCT/US2011/038710, filed Jun. 1, 2011,which claims the benefit of and priority to U.S. Provisional ApplicationNo. 61/350,303, filed Jun. 1, 2010, the disclosures of which are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Umbilical cord tissue is a rich source of stem cells. Blood from theumbilical cord includes stem cells, including hematopoietic stem cellsthat can be used to repopulate a person's blood and immune system.Wharton's Jelly, a gelatinous substance within the umbilical cord,contains an additional population of stem cells, distinct from thosefound in cord blood. As used herein, “Wharton's Jelly” can furtherinclude the amniotic epithelial layer of the umbilical cord. Processingand culturing the Wharton's Jelly permits the isolation of mesenchymalstem cells that can be used to regenerate a variety of tissues (see, forexample, U.S. Pat. No. 5,919,702).

SUMMARY OF THE INVENTION

The present inventors have discovered that the process of culturingcells from Wharton's Jelly substantially changes the characteristics ofthe cells. Compared to a population of cells cultured in vitro,uncultured Wharton's Jelly cells are molecularly different as can beseen, for example, in a different molecular profile on their cellsurfaces. More importantly, the inventors have found that minimallymanipulated Wharton's Jelly cells are substantially more potent in vivothan are cultured Wharton's Jelly cells.

The inventors have developed a method for purifying stem cells fromWharton's Jelly without the need for a culturing step. The methodincludes separating native, noncultured Wharton's Jelly stem cells froma digested tissue that includes Wharton's Jelly. The method can alsoinclude a prior step of digesting the tissue, for example bymechanically mincing the tissue or exposing it to a chemical or anenzyme such as a protease, for example, a collagenase, a hyaluronidase,or a dispase, separately or in combination. In addition, the method caninclude mechanically increasing the surface area of the tissue, such asby cutting or mincing the tissue, tearing it into small strands ormicroscopic pieces, or otherwise physically manipulating the tissue,prior to chemical or enzymatic digestion of the tissue. Furthermore,prior to digesting the tissue that includes Wharton's Jelly, the tissueis optionally dissected to remove arteries and veins.

Incomplete digestion can leave fragments of undigested tissue. Themethod can include separating the digested and undigested tissue, suchas by sedimenting the undigested tissue. The sedimentation process canbe accelerated by, for example, centrifugation. Alternatively, thedigested tissue can be filtered to remove the undigested tissue; thenoncultured Wharton's Jelly stem cells can be separated from thefiltrate, such as by sedimentation or filtration. In some embodiments,the digested tissue, which can be viscous, is washed or diluted before aseparating step, although other steps such as vigorous centrifugationcan be effective even in the absence of a washing or diluting step.

The inventors have also developed methods of recovering both culturedand noncultured stem cells from Wharton's Jelly. The method includespurifying noncultured Wharton's Jelly stem cells according to any of themethods described above, and culturing mesenchymal stem cells from theundigested tissue. In this manner, the uncultured stem cells of superiorpotency are obtained from the digested tissue and additional cells arecultured from the remnants of undigested tissue. The mesenchymal stemcells are optionally cultured in a medium that includes Wharton's Jelly.

The invention also relates to the purified, noncultured Wharton's Jellystem cells and their use. As used herein, “purified” indicates that theWharton's Jelly stem cells have been isolated and separated from certainacellular components of Wharton's Jelly, but does not indicate that thestem cells have necessarily been purified from other cell types that mayalso be present in Wharton's Jelly. In some embodiments, the purified,noncultured Wharton's Jelly stem cells are substantially free ofsemi-solid Wharton's Jelly. Some degree of liquefied Wharton's Jelly(digested into a viscous liquid, for example) may remain, or the cellsmay be entirely free of Wharton's Jelly and optionally in anothermedium, such as a sterile solution, a balanced salt solution, acryoprotectant solution, plasma, etc. In other embodiments, the purifiedWharton's Jelly stem cells are maintained at a temperature below 0° C.,below −20° C., below −80° C., or below −180° C., for example in a vial,bag, or other container suitable at such a temperature. The purified,noncultured Wharton's Jelly stem cells of the invention can differsubstantially from mesenchymal stem cells cultured from Wharton's Jelly,including differences in the level of cell surface expression of one (ortwo or three or four or more) of CD49B, CD105, CD133, HLA-ABC, CD73,CD44, SSEA-4, CD29, and/or CD90. In some embodiments, for example, thepopulation of noncultured Wharton's Jelly stem cells has reduced levelsof CD73 and CD 105 cell surface expression compared to mesenchymal stemcells cultured from Wharton's Jelly. Both CD73 and CD105 have beenreported to be markers for mesenchymal stem cells. Accordingly, thereduced level of CD73 and CD105 on the cell surfaces of nonculturedWharton's Jelly stem cells is consistent with the identification ofthese cells as substantially different from cultured mesenchymal stemcells.

The uncultured Wharton's Jelly stem cells are multipotent and can beadministered to a subject as a part of a therapeutic method, forexample, to heal a tissue or to assist in tissue regeneration. Incertain embodiments, the Wharton's Jelly stem cells are advantageouslyautologous or allogenic to the subject.

The invention also provides a homogenous solution including Wharton'sJelly. The solution can be obtained, for example, by digesting theWharton's Jelly to render it a viscous liquid and purifyingparticulates, such as undigested tissue or cells, from the digest toobtain a homogeneous solution. The solution can optionally be diluted,such as by a balanced salt solution or other sterile solution, to reduceviscosity. The solution can be depleted of cells, either by removingsubstantially all of the cells or by otherwise reducing the number ofcells in the solution. The solution can be frozen (for example, at atemperature of −20° C. or below), and can optionally be used in a cellculture process. Thus, the invention also provides methods ofmaintaining a cell by mixing the cell in a homogeneous solutionincluding Wharton's Jelly, for example by adding the cell to thesolution; by adding the solution to a suspension comprising the cell; orby applying the solution to a surface to which the cell has adhered. Thecell can be cultured in vitro. In one embodiment, the cell is amultipotent stem cell, such as a mesenchymal stem cell from Wharton'sJelly.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the present invention, as well as theinvention itself, can be more fully understood from the followingdescription of the various embodiments, when read together with theaccompanying drawings, in which:

FIG. 1 graphically depicts the in vivo efficacy of noncultured Wharton'sJelly stem cells (WJ MSCs) or culture-expanded mesenchymal stem cells(Expanded WJ MSCs) in a co-transplantation assay with humanhematopoietic stem cells from umbilical cord blood, with results shownas a % of bone marrow cells expressing human CD45 on their surface,which serves as a surrogate marker for engraftment of human cellstransplanted into mice;

FIG. 2 graphically depicts, for each of thirteen cell surface markers(CD49B, CD105, CD34, CD45, CD133, HLA-ABC, CD73, HLA-DR, CD14, CD44,SSEA-4, CD29 and CD90), in two-dimensions the number of nonculturedWharton's Jelly stem cells measured to have a particular expressionlevel of the marker on the cell surface; and

FIG. 3 is a comparable graphical depiction of the number ofculture-expanded mesenchymal stem cells from Wharton's Jelly having aparticular expression level of the marker on the cell surface.

DETAILED DESCRIPTION

The present application provides methods for purifying Wharton's Jellystem cells without the need for a culturing step. The resulting cellsare particularly useful therapeutically, having superior potency whencompared to stem cells expanded in culture from Wharton's Jelly. Theapplication also provides a homogeneous solution from Wharton's Jellythat can be used, for example, in a process of maintaining cells, suchas in culture.

To provide an overall understanding of the invention, certainillustrative embodiments will now be described. However, it will beunderstood by one of ordinary skill in the art that the compositions andmethods described herein may be adapted and modified as appropriate andmay be employed in other suitable applications. All such adaptations andmodifications are to be considered within the scope of the invention.

Purification of Noncultured Wharton's Jelly Stem Cells

The purification of the Wharton's Jelly stem cells requires separatingthe noncultured cells from a digested tissue that includes Wharton'sJelly. The digested tissue can be processed chemically. For example, thedigested tissue may result from enzymatic digestion of umbilical cordtissue, such as by a collagenase and/or another protease, such as ahyaluronidase and/or a dispase. In another example, tissue digestion maybe facilitated by acids. Optionally, an umbilical cord tissue can bedissected to remove the arteries and veins, and then processed tomaximize the available surface area. This processing can generallyinvolve any manner of mechanically increasing the surface area of thetissue, but most often involves finely cutting or microscopicallymincing the tissue into small strands or microscopic pieces, such aswith dissecting scissors or a scalpel.

Before the cells are separated from the digested tissue, any remainingfragments of undigested tissue are optionally discarded to facilitatethe subsequent purification of the cells. Depending on their size,undigested tissue could be removed by physical extraction (e.g. withforceps), decanting, aspiration, sedimentation (optionally acceleratedby centrifugation), or filtering, for example.

The separation of the cells from the digested tissue can be accomplishedby sedimentation of the cells from a homogeneous mixture containing thedigested tissue. Although gravity sedimentation can be used, thesedimentation process can be accelerated by, for example, a centrifugeto enhance the downward movement of the cells through (and, in somesense, out of) the mixture. One such process is described in Example 1.Upon separation, the Wharton's Jelly stem cells are substantially freeof the Wharton's Jelly. Because the digested tissue is generallyviscous, the tissue can be washed or diluted with an appropriate sterilesolution (such as a buffered salt solution) at any stage in the process.In fact, after the cells have been separated from the mixture, furtherwashes can be performed to further cleanse the cells as desired.

Native Wharton's Jelly Stem Cells

The purified, noncultured Wharton's Jelly stem cells can be usedimmediately in a patient, if there is an immediate need. Typically,however, the cells are cryopreserved in liquid nitrogen until needed,typically with a cryprotectant such as DMSO or dextran, and often in asolution such as autologous plasma or 5% human serum albumin. Asmultipotent stem cells, the noncultured Wharton's Jelly stem cells canbe used to treat or regenerate any of a variety of tissues such as bone,cartilage, fat or muscle. These cells can facilitate hematopoieticengraftment and have the potential to regulate and suppress immuneresponses in the host.

As described in Example 4, a population of purified noncultured cellsfrom Wharton's Jelly is demonstrably different, at the molecular level,from a population of cells from Wharton's Jelly that have been expandedin culture ex vivo. For example, although both populations include cellsexpressing CD49B, CD105, HLA-ABC, CD73, CD44, SSEA-4, CD29, and CD90,the populations differ in their expression profiles for most if not allof these markers. Thus, these or other markers can be used, individuallyor in combination (such as any two, three, four, five, six, seven, orall eight of these markers), to identify and characterize a populationof stem cells, such as those derived from umbilical cord tissue, and/orto characterize the biological potency of the cells.

Additional Products

The purification process also typically yields additional usefulproducts. For example, when the cells are separated from the digestedtissue, the remaining, cell-depleted digested tissue is a rich, sterilesolution that can be used for maintaining cells (in culture, forexample). It is appreciated that some cells may be present, although insubstantially reduced numbers, within this rich, sterile, cell-depletedsolution derived from the digested tissue. Alternatively, the solutionmay be completely devoid of cells. This homogeneous solution can befrozen (for example, at −20° C. or below) for later use.

Any fragments of undigested tissue remaining after a digestion processare also particularly useful, as these can be used as a source ofcultured mesenchymal stem cells using standard methods for expandingmesenchymal stem cells in culture from Wharton's Jelly. In fact, thehomogeneous Wharton's Jelly solution that is, in some sense, a byproductof the purification process can be used in the culturing of mesenchymalstem cells from the undigested tissue fragments. In this way, thepurification process whose primary purpose is the preparation ofnoncultured Wharton's Jelly stem cells can also provide, as an addedbenefit, mesenchymal stem cells that are expanded in culture from theundigested tissue fragments with the help of the cell-depleted Wharton'sJelly solution.

Accordingly, this invention provides two sources for the seeding andderivation of culture expanded Wharton's Jelly derived mesenchymal stemcells. The first source is the undigested umbilical cord tissue. Becauseenzymatic digestion rarely digests the tissue completely, the remainingundigested tissue can be utilized as a seeding source for the expansionof mesenchymal stem cells. A second source for the derivation ofmesenchymal stem cells is the Wharton's Jelly stem cells derived fromthe digested tissue. Enzymatic digestion cleaves collagen cross-linkswithin the Wharton's Jelly and releases the embedded cells. As describedpreviously, the released cells in the form of single-cell suspensionscan be processed and cryopreserved for later therapeutic use.Alternatively, these cells can be used as a seeding source for theexpansion of mesenchymal stem cells. In addition, the post-digestioncell-depleted Wharton's Jelly can be used as a supplement for thederivation of mesenchymal stem cells from both digested and undigestedtissues. Its use as a supplement is not necessary for culture derivationbut may reduce the time required for derivation and expansion.

In addition to the two-dimensional adherence culture routinely used forthe expansion of mesenchymal stem cells, bioreactors may be used toexpand mesenchymal stem cells in three-dimensional suspension cultures.Bioreactors allow for scaled-up production and closely mimic the in vivoperfusion characteristics of the umbilical cord. Mesenchymal stem cellscan be derived initially from either two-dimensional orthree-dimensional cultures and subsequently propagated inthree-dimensional cultures for scaled-up production. The bioreactors maybe supplemented with microcarrier beads to enable the adherence andpropagation of the mesenchymal stem cells within the bioreactors.

EXAMPLES

The invention is further illustrated by the following examples, whichare provided for illustrative purposes only, and should not be construedas limiting the scope or content of the invention in any way.

Example 1 Purification of Native Wharton's Jelly Stem Cells andHomogeneous Wharton's Jelly Solution

Umbilical cords were collected in sterile specimen containers within 48hours of the time of delivery. In a biosafety cabinet, 10 mL of Buffer B(50 μg/mL gentamicin, 100 units/mL penicillin and 100 μg/mL streptomycinin sterile Dulbecco's phosphate buffered saline) were added to theumbilical cord in an umbilical cord collection chamber. Otherantibiotics, such as 0.25 μg/mL amphotericin B, 100 μg/mL streptomycinand/or 10 μg/mL ciprofloxacin, can also be added to Buffer B orsubstituted for any of the antibiotics in Buffer B. The contents of thecollection chamber were then mixed by swirling and maintained at roomtemperature for fewer than 72 hours. The contents were swirled again forapproximately 10-15 seconds to clean the umbilical cord tissue.Coagulated blood, if evident on the surface of the umbilical cord, wascarefully removed using dissection tools.

The umbilical cord was transferred to a Petri dish using sterile forcepsand cut into 3-5 cm segments using a sterile umbilical cord scissor orscalpel. Each segment was then individually dissected as follows.Briefly, a segment to be dissected was placed on a 150 mm Petri dish.The two arteries and one vein of the umbilical cord were located byviewing the cross-section of the tissue segment. Using dissectingscissors, an incision was made between the two arteries. With two tissueor Dumont forceps, the cord was pulled apart along the length of thetissue, carefully tearing the tissue away from the arteries and thevein. Once the tissue was opened, the vein was located and excised usinga sterile fine point forceps in each hand. The two arteries weresubsequently located and excised, and the dissected tissue was placed onthe sterile, inside lid of the 150 mm dissection plate.

The tissue was then minced into small pieces/strands with dissectingscissors for at least 5 minutes or until a consistent minced tissue wasobtained. The initial dissection and mincing were performed on differentportions of the dissection plate to minimize contamination withexcessive blood and/or dissected vessels. The final minced tissue lookedlike ground tissue and had no or few obvious tissue chunks. Generally,the minced tissue pieces had a cross-section of about 1 mm². The mincedtissue was placed in a sterile, labeled conical tube.

Once all of the umbilical cord tissue segments were dissected, mincedand added to the conical tube, 10 mL of solution CB (2.5 mg/mLcollagenase NB6 (Serva) and 2 mM calcium chloride in Dulbecco'sphosphate buffered saline) were also added. The contents of the tubewere mixed by inverting/shaking several times until a uniform mixturewas obtained. Parafilm was placed around the tube cap to prevent leakageand cross-contamination. The outside of the tube was sprayed with 70%ethanol and placed on an orbital shaker/mixer at approximately 175 RPMinside a 37° C. incubator for approximately two hours. Every hour thetube was shaken vigorously to help further dissociate the tissue. Afterapproximately two hours, the tube was again sprayed with 70% ethanol andreturned to the biosafety cabinet.

The digested tissue was then filtered using a Steriflip® filter unit(Millipore) to remove any undigested tissue from the digested tissue. Asthe digested Wharton's Jelly has a viscous, ‘honey-like’ consistency,care was taken to prevent contamination when opening caps and handlingthe jelly. The tube was placed upright, and its cap was removedcarefully. To remove the jelly connecting the tube and the cap ascompletely and sterilely as possible, the tube and cap were pulled apartuntil small strands of the jelly followed. Circular motions then removedthe final jelly strands from the cap. Care was taken not to contaminatethe neck of the tube with the jelly. Once the cap was removed, 20 mL ofDulbecco's phosphate buffered saline were added to dilute the jelly. Thecap was replaced and tightened, and the tube was inverted or shakenseveral times to mix. The cap was again removed carefully and theSteriflip® filter unit was screwed on the top of the tube and tightlysecured. The assembly was then flipped over so that the 50 mL conicaltube was upside down.

A regulated vacuum source was attached to the vacuum port on the side ofthe filter unit. The filter unit was maintained in an upright positionwhile filtering. If needed, the tube/filter assembly was swirledvertically to dislodge tissue trapped in the filter. Once all liquid wasfiltered, the vacuum was shut off and the 50 mL conical tube wasremoved. 10 mL of Dulbecco's phosphate buffered saline were added to the50 mL conical tube to wash any remaining cells that may have adhered tothe sides or bottom of the tube. The cap of the tube was replaced andtightened and the tube was inverted or shaken several times to wash thebottom and sides of the tube. The cap was again removed and the tubeagain attached to the filter unit. The vacuum was reapplied until allliquid had passed through the filter, at which point the assembly wasdisconnected from the vacuum.

The total volume of the filtrate was approximately 50 mL. If needed,Dulbecco's phosphate buffered saline was added to the filtrate to bringthe final volume to 50 mL. The cap was placed on the filtrate tube andtightly secured. The outside of the tube was sprayed with 70% alcoholand sealed with film to prevent leakage and cross-contamination. Thetube was inverted/shaken several times until the jelly and theDulbecco's phosphate buffered saline were homogenized. The tube was thenplaced on a shaker (at 175 RPM) in a 37° C. incubator for five minutesto further homogenize the jelly, with additional inversion/shaking asneeded until a uniform mixture was obtained. The tube was again sprayedwith 70% ethanol and returned to the biosafety cabinet.

The homogenized, digested Wharton's Jelly was then split into a numberof 50 mL conical tubes depending on the initial weight of the umbilicalcord. If the initial weight was no more than 15 grams, a single conicaltube was used. If the initial weight was no more than 30 grams, twotubes were used. The number of tubes used was equal to the initialweight of the umbilical cord in grams, divided by 15, rounded up. Eachtube received an approximately equal volume of the jelly; care was takennot to contaminate the necks of the tubes.

The volume of each tube was then brought to 50 mL with Dulbecco'sphosphate buffered saline, and the contents of each tube were againhomogenized. Subsequently, each tube was tightly capped; sprayed with70% ethanol; sealed with film; inverted/shaken a number of times; andplaced on a shaker (at 175 RPM) in a 37° C. incubator for five minutesto further homogenize the jelly, with additional inversion/shaking asneeded until a uniform mixture was obtained.

Once homogenized, the tubes were spun for 20 minutes at 750×g at 37° C.After spinning a cell pellet was normally present at the bottom of each50 mL tube. In the absence of a pellet, the tubes were respun at 1000×gfor 15 minutes at 37° C. The tubes were sprayed with 70% ethanol andreturned to the biosafety cabinet. The supernatant was decanted slowly,at a constant rate, without shaking or rocking the tube to avoiddislodging the pellet. The decanted supernatant, a homogenous Wharton'sJelly solution depleted of cells, was stored at or below −20° C. as aseparate reagent useful for culturing stem cells.

To each cell pellet, 10 mL of Dulbecco's phosphate buffered saline wereadded. The tubes were securely capped and were vortexed, inverted,and/or pipetted several times to mix well until the cells werecompletely suspended. The contents of all sample tubes were thencombined using a pipette into one tube. The sample tubes were rewashedwith Dulbecco's phosphate buffered saline to dislodge any remainingcells, which were also added to the combined tube, and the volume wasbrought to 50 mL using Dulbecco's phosphate buffered saline. The tubewas capped and vortexed/inverted several times to mix. The tube was spunfor 15 minutes at 500×g at 37° C. A cell pellet was normally present atthe bottom of the tube. In the absence of a pellet, the tube was respunat 750×g for 10 minutes at 37° C. The supernatant was carefully decantedinto a waste flask so as not to disturb the cell pellet.

Subsequently, 25 mL of Dulbecco's phosphate buffered saline were addedto the cell pellet and, after the tube was capped and vortexed/invertedseveral times to resuspend the cells, the cells were passed through a 70micron tube-top filter. The filter was placed on top of a sterile 50 mLconical tube. The resuspended cells were released drop-wise from asterile 25 mL pipette, directly above the center of the filter but nottouching the filter. The filtered cell suspension collected in the 50 mLconical tube. An additional 20 mL of Dulbecco's phosphate bufferedsaline were used to wash the previous tube to maximize cell recovery;after washing the tube, these 20 mL were also passed dropwise throughthe filter. Additional Dulbecco's phosphate buffered saline was passeddropwise through the filter to bring the final volume to 50 mL.

The filtrate was spun for 10 minutes at 500×g at 37° C. A cell pelletwas normally present at the bottom of the tube after the spin. In theabsence of a pellet, the tube was respun at 750×g for 10 minutes at 37°C. The outside of the tube was sprayed with 70% alcohol before returningto the biosafety cabinet, where the supernatant was decanted off into awaste flask carefully, so as not to disturb the cell pellet. The volumein the tube was brought up to 4.3 mL using Dulbecco's phosphate bufferedsaline and the contents of the tube were mixed by pipetting, shaking,and/or vortexing. With a 1000 μL pipette, the cell suspension was mixedand a 0.3 mL aliquot was removed for quality control analysis, leaving4.0 mL of a purified cell suspension of noncultured Wharton's Jelly stemcells.

Example 2 Storage of Noncultured Wharton's Jelly Stem Cells

The purified cell suspension of Example 1 was cryopreserved in a 25 mLfreezing bag. Using a 60 mL syringe with an 18G needle, 16 mL ofautologous plasma, 5% human serum albumin, or a combination thereof wereadded to the 4 mL purified Wharton's Jelly stem cell suspension. Analcohol pad was used to wipe the top of a vial of 55% DMSO/5% Dextran.Next, 5 mL of the DMSO/Dextran mixture were removed using a 60 mLsyringe with an 18G needle and slowly added to the cell suspension. Thecell suspension tube was capped tightly and gently inverted to mix,taking care not to make foam or bubbles. Using the same 60 mL syringe,25 mL of the cell suspension were transferred to the freezing bag. Thefreezing bag was stored in a metal canister in a Styrofoam holder at−80° C. for 16 to 24 hours, optionally followed by an intervening periodin a liquid nitrogen freezer in which the cells were exposed only to thevapor phase of the liquid nitrogen and, ultimately, in the liquid phaseof liquid nitrogen for permanent storage. Alternatively, the freezingbag with cells can be permanently stored in the vapor phase of theliquid nitrogen.

Example 3 In Vivo Efficacy

The therapeutic efficacy of noncultured Wharton's Jelly stem cells wasdemonstrated in a co-transplantation assay with hematopoietic stem cellsfrom umbilical cord blood to renew a mammalian hematopoietic system.

Hematopoietic stem cells from umbilical cord blood can be administeredto a mammal to reconstitute a hematopoietic system damaged, for example,by radiation. Co-transplantation of Wharton's Jelly stem cells improvesthe reconstitution process, enhancing the engraftment of theadministered hematopoietic stem cells and, therefore, their ability toproliferate and recreate a hematopoietic system in their new host.

To test the efficacy of noncultured Wharton's Jelly stem cells, theywere co-administered with hematopoietic stem cells from umbilical cordblood to (NOD/SCID IL2Rλ-null) mice that had been sublethally irradiatedthe day before with 300 cGy of gamma-radiation which ablated the bonemarrow. 1,000,000 mononuclear umbilical cord blood cells wereadministered to the mice via the tail vein, either alone or with 10,000,50,000, or 100,000 noncultured Wharton's Jelly stem cells, or with1,000,000 mesenchymal stem cells cultured from Wharton's Jelly. Sixtydays later, the bone marrow was obtained from the mice to measure thenumber of cells expressing human CD45, a cell surface marker for humanhematopoietic cells and a surrogate marker for human hematopoietic stemcell engraftment.

As shown in FIG. 1, irradiated mice which did not receive any cellslacked human CD45-expressing cells in their bone marrow on day 60.Although mice receiving only mononuclear umbilical cord blood cellsshowed a substantial number of bone marrow cells expressing human CD45,this number more than tripled in mice that received mesenchymal stemcells cultured from Wharton's Jelly or noncultured Wharton's Jelly stemcells. Surprisingly, 100,000 noncultured Wharton's Jelly stem cellsprovided a benefit equal to or greater than the benefit of 1,000,000culture-expanded mesenchymal stem cells from Wharton's Jelly, suggestingthat noncultured Wharton's Jelly stem cells may be more than ten-foldmore potent in vivo than cultured mesenchymal stem cells. In fact, asfew as 10,000 noncultured Wharton's Jelly stem cells were nearly aseffective as 1,000,000 cultured mesenchymal stem cells. While theprecise mechanism for the reduced efficacy of cultured mesenchymal stemcells is unclear, minimally manipulated, uncultured Wharton's Jelly stemcells appear to have important therapeutic advantages in vivo.

Example 4 Differences in Cell Surface Marker Profiles

Noncultured Wharton's Jelly stem cells are also noticeably different atthe molecular level from mesenchymal stem cells cultured from Wharton'sJelly.

The levels of thirteen cell surface markers on a population ofnoncultured Wharton's Jelly stem cells were assayed by standardantibody/flow cytometry assays, and the results are depicted in FIG. 2.FIG. 2 provides, for each marker tested, a standard two-dimensionalrepresentation showing the percentage of cells demonstrating aparticular expression level of the marker, as detected by antibodyassay. The results from the noncultured Wharton's Jelly stem cells arerepresented with a dark line, and the results from a control arerepresented with a lighter line. As shown, noncultured Wharton's Jellystem cells exhibited higher levels of CD49B, CD105, HLA-ABC, CD73, CD44,SSEA-4, CD29, and CD90 compared to the antigen control. NonculturedWharton's Jelly stem cells did not demonstrate substantial expression ofCD34 and CD45, markers that would be typical of hematopoietic cells, orof CD14, HLA-DR or CD133.

The expression of the same markers was tested in mesenchymal stem cellscultured from Wharton's Jelly; the results are depicted in FIG. 3. Asshown, compared to the antigen control, cultured mesenchymal stem cellsdemonstrated higher levels of CD49B, CD105, HLA-ABC, CD44, CD29, CD73and CD90; a broader range of levels of expression of SSEA-4; and a lackof CD14, CD34, CD45, and HLA-DR. Comparing FIG. 2 and FIG. 3, even formarkers such as CD105 which are elevated both in cultured mesenchymalstem cells and in noncultured Wharton's Jelly stem cells, the patternsof expression can be very different, as the observed levels ofexpression in noncultured Wharton's Jelly stem cells substantiallyoverlap with the antigen control, whereas the mesenchymal stem cellscultured ex vivo show much higher levels of expression with littleoverlap with the antigen control. Thus, cells that have been purifiedfrom Wharton's Jelly without culturing are not merely particularlypotent in vivo, they are also markedly different from mesenchymal stemcells cultured and expanded from Wharton's Jelly as evidenced by theirpatterns of cell surface markers.

INCORPORATION BY REFERENCE

The entire disclosures of each of the patent documents and scientificarticles cited herein are incorporated by reference in their entiretyfor all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

We claim: 1-30. (canceled)
 31. A cell culture medium comprising acell-depleted Wharton's Jelly filtrate, wherein the filtrate is preparedby a method comprising mincing umbilical cord tissue comprisingWharton's Jelly, subsequently diluting the umbilical cord tissue,subsequently filtering the umbilical cord tissue to generate a filtrate,and sedimenting Wharton's Jelly stem cells from the filtrate.
 32. Amethod of culturing mesenchymal stem cells, the method comprising: (a)culturing mesenchymal stem cells in a medium comprising a cell-depletedWharton's Jelly filtrate, wherein the filtrate was previously preparedby mincing umbilical cord tissue comprising Wharton's Jelly,subsequently diluting the umbilical cord tissue, subsequently filteringthe umbilical cord tissue to generate a filtrate, and sedimentingWharton's Jelly stem cells from the filtrate.
 33. A method of culturingmesenchymal stem cells, the method comprising: (a) mincing umbilicalcord tissue comprising Wharton's Jelly; (b) subsequently diluting theumbilical cord tissue; (c) subsequently filtering the umbilical cordtissue to generate a filtrate; (d) sedimenting Wharton's Jelly stemcells from the filtrate; and (e) culturing mesenchymal stem cells in amedium comprising the filtrate.